The long-term objective of this study is to increase our understanding of the use of conserved membrane proteins as components of a vaccine for prevention of Neisseria meningitidis serogroup B (MenB) disease. MenB is a major cause of meningitis and sepsis. Although serum bactericidal antibodies confer protection, to date, conventional approaches to develop a vaccine have been largely unsuccessful. Polysaccharide-based MenB vaccines risk eliciting autoantibodies to host polysialic acid, while the ability of most non-capsular antigens to elicit broad-based immunity is limited by antigenic diversity. We propose to investigate the vaccine potential of three recently discovered conserved Neisserial membrane proteins, designated Neisserial surface proteins (Nsp) A, B, and C. As backup candidates, NspD and NspE are also available. NspA was discovered with a monoclonal antibody, while the other four proteins represent new vaccine candidates that were discovered from analysis of genomic data. All five proteins are highly conserved across pathogenic Neisseria, have epitopes on the surface of the bacteria that are accessible to antibody, and elicit complement-mediated bactericidal antibodies in mice or rabbits. Thus, each of these proteins deserves further investigation as candidate antigens for inclusion in a MenB vaccine. In Aim 1, we will investigate the immunogenicity of each of the recombinant proteins in mice and guinea pigs. Should the recombinant molecules fail to elicit high titers of antibodies that are functionally active against the bacteria, we will attempt to reconstitute conformational epitopes with the use of detergents or liposomes, and explore the use of novel adjuvants suitable for human use. In Aim 2, we will prepare monoclonal antibodies (Mabs) that react with epitopes on the Ns proteins that are important in eliciting protective antibodies. These Mabs will be used for epitope mapping, and for studies of antibody functional activity. In Aim 3, we also will use the 3Iabs to investigate whether there are strain differences in surface accessibility and expression of the different NS proteins, and correlate any differences found with the respective DNA sequences encoding the proteins, or transcriptional activity of the respective genes. We also will investigate whether surface accessibility of the different Ns proteins varies within a Neisserial strain when propagated in vitro, or in infant rats. In Aim 4, we will test the hypothesis that a vaccine containing more than one Ns protein will elicit broader protective immunity to MenB than a vaccine made from a single protein. These results are directly relevant to evaluating the potential for inclusion or exclusion of each of these novel proteins in a MenB vaccine. The data also may validate the genomic approach for identification of new antigenic targets for vaccine development.